Insect cell
Recombinant protein expression using the baculovirus expression system. Since the first recombinant protein productions using a baculovirus vector were reported about 40 years ago, the baculovirus expression vector system (BEVS) has become one of the most popular eukaryotic systems for recombinant protein production for research purposes.
The BEVS is used both for the expression of intracellular and extracellular (secreted) proteins as well as membrane proteins, its particular strength can be described with these highlights:
1. Intracellular/secreted proteins with post-translational modifications (PTMs): It allows PTMs similar or resembling those in mammalian cells [1-3]. It is therefore popular for the expression of intracellular mammalian protein kinases, a major drug target. However, glycoproteins produced in insect cells show more uniform, but less complex N-glycans than those produced in mammalian cells [4].
2. Membrane proteins: It is very powerful in the field of membrane structural biology. Approximately 35% of currently approved drugs, target one class of membrane proteins, the G protein-coupled receptors (GPCRs). 85% of the deposited structures [5] of all GPCR structures deposited in the Protein Data Bank have been expressed using the BEVS.
3. Multiprotein complexes: The Baculovirus genome can incorporate large inserts, a fact that has been exploited to develop platforms to produce multi-protein complexes from one baculovirus [6]. It should be noted that the BEVS also offers the possibility to infect cells for protein expression with two or more viruses simultaneously.
LP3 at Lund University, now part of PPS, has used the BEVS to provide recombinant proteins to its users since 2013 and collaborates both in the development and implementation of “best practice” for the BEVS with other protein production platforms internationally and disseminates knowledge about use of the system [7, 8].
The main steps in recombinant protein production using the BEVS can be described as (Figure 1):
A. Design and generation of a donor plasmid for the BEVS
B. Bacmid generation and verification
C. Transfection and Baculovirus Infected Insect Cell Stock Creation (BIIC)
D. Test expressions
E. Large scale expression
For more information and detailed protocol descriptions please see here [7]
Beyond the production of recombinant proteins as single or multiprotein complexes other areas of use of the BEVS can be described under these main categories [2]:
1. Surface display and baculovirus particles as antigen carriers
2. Production of heterologous viral vectors in insect cells
3. Baculovirus-based gene delivery vehicles for mammalian cells
What PPS offers:
We provide recombinant proteins expressed in insect cells for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. We can assist with the whole process of protein production or any particular step in the process outlined above:
A. Donor plasmid design with codon optimized and tagged insert
B. Bacmid generation and verification
C. Transfection and Baculovirus Infected Insect Cell Stock Creation (BIIC)
D. Test expressions with Spodoptera frugiperda (Sf9) or Trichoplusia ni (High Five) cells
E. Large-scale expressions from 1 to 24 L (6 L as standard)
Figure 1.
Main steps in recombinant protein production using the BEVS
[1] Chen N, Kong X, Zhao S, Xiaofeng W. Post-translational modification of baculovirus-encoded proteins. Virus Res. 2020;279:197865.
[2] van Oers MM, Pijlman GP, Vlak JM. Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology. J Gen Virol. 2015;96:6-23.
[3] McKenzie EA, Abbott WM. Expression of recombinant proteins in insect and mammalian cells. Methods. 2018;147:40-9.
[4] Harrison RL, Jarvis DL. Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce "mammalianized" recombinant glycoproteins. Adv Virus Res. 2006;68:159-91.
[5] Errey JC, Fiez-Vandal C. Production of membrane proteins in industry: The example of GPCRs. Protein Expr Purif. 2020;169:105569.
[6] Gorda B, Toelzer C, Aulicino F, Berger I. The MultiBac BEVS: Basics, applications, performance and recent developments. Methods Enzymol. 2021;660:129-54.
[7] Sullivan HM, Krupinska E, Rasmussen AA, Orozco Rodriguez JM, Knecht W. Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS). Methods Mol Biol. 2023;2652:55-77.
[8] Stolt-Bergner P, Benda C, Bergbrede T, Besir H, Celie PHN, Chang C, et al. Baculovirus-driven protein expression in insect cells: A benchmarking study. J Struct Biol. 2018;203:71-80.
